The overuse of antibiotics and the prescription of first-line antibiotics to which a pathogen is not susceptible, contribute to rising antibiotic resistance rates, which is a growing threat to public health worldwide.1 Urinary tract infections are among the most prevalent bacterial infections.2 Gold-standard antibiotic susceptibility tests for urinary tract infections rely on culture and typically require 1-3 days in order to allow the bacteria to multiply to detectable levels.3 After pre-culture of the bacteria, an additional 18 hours are typically required to perform standard susceptibility tests. Reducing the time needed to determine the susceptibility profile of urinary tract infections could improve clinical outcomes, especially in the case of the most severe infections that lead to urosepsis.4 Rapid testing could also contribute to decreased unnecessary antibiotic use,5 and could increase the efficiency of centralized diagnostic laboratories. Treatment of other infections may similarly benefit from improved susceptibility testing.
Tests for antibiotic resistance that rely on enzymatic amplification of antibiotic-resistance genes have been found to reduce turnaround times compared to culture.6,7,8,9 Unfortunately, these assays often require a pre-incubation step to allow the bacteria to multiply, and, further, often require several hours to amplify the genes of interest. Gene-based assays are also typically limited by the requirement of knowing a priori which genes confer resistance. Dozens of constantly-evolving genes may be implicated in resistance to a given antibiotic, and it may be impractical to test for all possible mutations simultaneously.10 
Assays that monitor bacterial viability in response to antibiotics may overcome at least some limitations of genetic tests. These tests report directly on the question of greatest clinical importance: whether a given antibiotic decreases bacterial survival. New assays for antibiotic resistance include the detection of bacterial motion using AFM cantilevers,11 electrochemical measurements of bacterial growth,12,13,14,15,16 optical detection of bacterialgrowth,17,18 and optical detection of redox reporters of bacterial metabolism.19,20,21,22 In assays that detect metabolically-active pathogens, the bacteria are typically incubated with the antibiotic and a redox reporter of metabolism such as resazurin or methylene blue. Metabolically-active bacteria create a reducing environment and either directly or indirectly reduce the compound, and the change in redox state is read out as a change in color or fluorescence. Resistant bacteria continue to multiply and metabolize the compound, while susceptible bacteria do not.
Successful detection using this type of approach typically hinges on the requirement that a sufficient quantity of the reduced form of the reporter compound accumulates above the detection threshold, a delay that may take at least 12 hours in milliliter-scale culture.19 Strategies have been proposed that seek to confine bacteria in microliter and nanoliter volumes with the goal of reducing the time of detection by increasing the local concentration of the bacteria.20,21,23,24,25 In the most sensitive of these optical techniques, the sample is divided into millions of nanoliter droplets and the signal is readout sequentially from each droplet with a high-powered fluorescence microscope.20,21,25 Despite the increase in local effective concentration provided by this approach, several hours are typically still required for analysis. Moreover, many of these devices only detect the presence or absence of a pathogen and not its antibiotic susceptibility profile.25,26,27 